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Affinity and Structural Analysis of the U1A RNA Recognition Motif with Engineered Methionines to Improve Experimental Phasing

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dc.contributor.author Lippa, Geoffrey
dc.contributor.author Srivastava, Yoshita
dc.contributor.author Bonn-Breach, Rachel
dc.contributor.author Chavali, Sai Shashank
dc.contributor.author Jenkins, Jermaine
dc.contributor.author Wedekind, Joseph
dc.date.accessioned 2021-07-14T20:07:00Z
dc.date.available 2021-07-14T20:07:00Z
dc.date.issued 2021-03
dc.identifier.citation Srivastava, Y.; Bonn-Breach, R.; Chavali, S.S.; Lippa, G.M.; Jenkins, J.L.; Wedekind, J.E. Affinity and Structural Analysis of the U1A RNA Recognition Motif with Engineered Methionines to Improve Experimental Phasing. Crystals 2021, 11, 273. https://doi.org/10.3390/cryst11030273 en_US
dc.identifier.uri http://hdl.handle.net/10829/24550
dc.description This article is published open access in Crystals, also available at https://doi.org/10.3390/cryst11030273. Distributed under the CC-BY 4.0 license. en_US
dc.description.abstract RNA plays a central role in all organisms and can fold into complex structures to orchestrate function. Visualization of such structures often requires crystallization, which can be a bottleneck in the structure-determination process. To promote crystallization, an RNA-recognition motif (RRM) of the U1A spliceosomal protein has been co-opted as a crystallization module. Specifically, the U1-snRNA hairpin II (hpII) single-stranded loop recognized by U1A can be transplanted into an RNA target to promote crystal contacts and to attain phase information via molecular replacement or anomalous diffraction methods using selenomethionine. Herein, we produced the F37M/F77M mutant of U1A to augment the phasing capability of this powerful crystallization module. Selenomethionine-substituted U1A(F37M/F77M) retains high affinity for hpII (KD of 59.7 ± 11.4 nM). The 2.20 Å resolution crystal structure reveals that the mutated sidechains make new S-π interactions in the hydrophobic core and are useful for single-wavelength anomalous diffraction. Crystals were also attained of U1A(F37M/F77M) in complex with a bacterial preQ1-II riboswitch. The F34M/F37M/F77M mutant was introduced similarly into a lab-evolved U1A variant (TBP6.9) that recognizes the internal bulged loop of HIV-1 TAR RNA. We envision that this short RNA sequence can be placed into non-essential duplex regions to promote crystallization and phasing of target RNAs. We show that selenomethionine-substituted TBP6.9(F34M/F37M/F77M) binds a TAR variant wherein the apical loop was replaced with a GNRA tetraloop (KD of 69.8 ± 2.9 nM), laying the groundwork for use of TBP6.9(F34M/F37M/F77M) as a crystallization module. These new tools are available to the research communit en_US
dc.language.iso en_US en_US
dc.publisher MDPI en_US
dc.relation.uri https://doi.org/10.3390/cryst11030273 en_US
dc.rights.uri https://creativecommons.org/licenses/by/4.0/ en_US
dc.title Affinity and Structural Analysis of the U1A RNA Recognition Motif with Engineered Methionines to Improve Experimental Phasing en_US
dc.type Journal Article en_US


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